PROJECT SUMMARY Most eukaryotic proteins that function in the endomembrane system or extracellular space traverse the protein- conducting translocon channel embedded in the endoplasmic reticulum (ER) membrane during or shortly after translation. Proteins that arrest during translocation prevent the passage of other proteins and impair cell function. Eukaryotes have evolved three proteolytic quality control (QC) mechanisms to eliminate proteins that arrest during translocation. In the first, ER-Associated Degradation of Translocon-associated proteins (ERAD- T), homologs of the ER-resident Hrd1 ubiquitin ligase (E3) catalyze destruction of proteins that arrest during translocation. Apolipoprotein B, the major protein component of mammalian low-density lipoproteins (LDL, i.e. ?bad cholesterol?), is targeted for degradation by the Hrd1 homolog gp78 if its translocation arrests. ERAD-T is strongly impaired during ER stress (an increased burden of unfolded proteins that is associated with certain cancers, neurodegenerative conditions, and immune disorders). In the second mechanism, ERAD of Ribosome- Associated proteins (ERAD-RA), the cytosolic E3 Rkr1/Ltn1 (in yeast) or listerin (in mammals) promotes destruction of proteins that simultaneously arrest in the ribosome and translocon. In the third mechanism, the protease Ste24 (in yeast) or ZMPSTE24 (in human cells) cleaves proteins engineered to clog the translocon. Consistent with the mission of the National Institute of General Medical Sciences, the objective of the proposed work is an improved understanding of translocon QC, an area of biology about which little is known, using yeast as a model system. Insights about translocon QC are likely to enhance the understanding of LDL physiology and the development of improved therapeutic strategies for cholesterol- and ER-stress-related pathologies. A yeast genomic screen recently revealed several genes with potential roles in ERAD-T. Among others, genes required for ER autophagy and other aspects of cellular QC were identified. The specific aims of this project are to (1) Characterize the roles of novel mediators and regulators of translocon QC and (2) Identify and characterize novel physiological substrates of translocon QC pathways. Beginning with genes with roles in ER autophagy and cellular QC, genes identified in the screen will be genetically and biochemically validated. Epistasis analysis will be conducted to determine if genes function with HRD1. The specificity of gene products for ERAD-T substrate degradation will be determined. Further, the ER stress sensitivity of gene product expression, localization, and function will be analyzed. Mechanistic experiments will elucidate specific roles of gene products in translocon QC. Finally, mass spectrometry will be performed to identify physiological translocon-associated substrates of each QC enzyme (Hrd1, Rkr1, and Ste24). Biological implications and mechanistic details of degradation of identified translocon-associated substrates will be investigated. This work will engage undergraduates and master?s students in biomedically relevant research. Students will participate in all aspects of this project, including experiment design, data collection, and communication of results.